Urothelial Cells UROtsa Exposed to or Malignantly Transformed by Cadmium or Arsenite Jennifer Larson Doctoral Candidate University of North Dakota October 5 2011 Outline Background Info ID: 337038
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The Expression of SPARC in Human Urothelial Cells (UROtsa) Exposed to or Malignantly Transformed by Cadmium or Arsenite
Jennifer Larson
Doctoral Candidate
University of North Dakota
October 5, 2011Slide2
OutlineBackground InfoPurposeMethods ResultsConclusionsFuture directionsSlide3
Bladder CancerTransitional cell carcinoma of the bladder is the 9th most common cancer worldwide and the 4th in U.S (Bischoff & Clark, 2010)
Highest cost per patient of all cancers
(Sullivan
et al.
, 2010; Jacobs
et al
., 2010)
Bladder cancer was the first cancer in which industrial carcinogens were found to play the major role in disease causation
Link between exposure to aromatic amines and development of bladder cancer in factory workers
(Rehn, 1895)
Cd⁺² and As⁺³ are human carcinogens
(IARC, 1993; IARC, 1980)
and linked to the development of bladder cancer
(Steinmaus
et al
., 1994 & 2000)
Association between cigarette smoking and bladder cancer
2-4 times increased risk
(Clavel
et al.,
1989; Morrison
et
al., 1984; Silverman
et al
., 1992)Slide4
UROtsa Cell LineDerived from the urothelium lining the ureter
of a 12 year-old female.
Immortalized with SV40 large T-antigen
(
Perzoldt
et al
., 1995)Cd⁺² and As⁺³ are able to cause the malignant transformation of UROtsa cells (Sens et al., 1994 & 2000)Generated 7 different Cd+2 & 6 different As+3 cell lines.
UROtsa parent cells are non-tumorigenic: Do not grow in soft agarNo not form tumors in nude mice
Transformed cell lines are tumorigenic:Grow in soft agarForm tumors in nude mice
UROtsa Parent
UROtsa Cd⁺²
UROtsa As⁺³
Sens et al.
Toxicological Sciences
(2004)Slide5
SPARC Secreted Protein, Acidic and
R
ich in
C
ysteine
Also known as: Osteonectin & BM-40
Belongs to the Matricellular group of proteinsSecreted macromolecules that interact with cell-surface receptors, ECM, and/or growth factors and proteases, but do NOT have structural rolesKnown to bind to structural matrix proteinsCollagen and VitronectinFound in tissues undergoing repair/remodeling (Salonen et al. 1990)Upregulated during embryological development (Sage et al., 1989)Other matricellular proteins include:Thrombosponbin 1 & 2Tenascin C & X
OsteopontinCTGFTestican 1, 2, & 3 (SPARC-related protein)Hevin (SPARC-related protein)Slide6
SPARC3 General functions of SPARC:1. De-adhesion: Exogenous SPARC inhibits cell spreading and cell attachment
(Sage et al. 1989)
Disassemble focal adhesions
(Murphy-Ullrich et al. 1995, Murphy-Ullrich 2001)
Reorganizes actin stress fibers to periphery of cell
(Murphy-Ullrich et al. 1995)
May play a role in cell migration
2. Anti-proliferation: Cell cycle inhibitor (Funk & Sage 1991, 1993; Sage et al. 1995)Promoted prostate cancer cell migration (De et al. 2003) and stimulated fibroblasts during myocardial infraction (Wu et al. 2006)May play a role in cell migration 3. Regulation of ECM & growth factors: Binds to many ECM components (Brekken & Sage 2000)Known activator of MMP-2 (Tremble et al. 1993)
Regulates PDGF, VEGF, & TGF-β1 expression (Raines et al, 1992, Kuppricon et al. 1998, Abe et al. 2004, Hasselaar & Sage 1992, Lane & Sage 1994)After exogenous treatment, capable of inducing the expression of SPARCMay play a role in the progression of metastatic tumors Slide7
PurposeTo determine the role SPARC plays in the formation and progression of bladder cancer.Slide8
SPARC Expression in UROtsa Cell Lines & Normal Bladder Tissue
Larson
et al
2010Slide9
SPARC Expression in Normal and Cancerous Bladder Tissue
Normal Bladder Tissue
High Grade Carcinoma
Larson
et al
2010Slide10
Possible role of epigentic modification?24, 48, and 72h treatment
48h shown
MS-275 is a
histone
deacetylase
inhibitor
Prevents acetyl groups from being removed from histones5-Aza-2’-deoxycytidine (5-AZC) is a methylation inhibitorInhibits the methylation of histonesConclusion: No change in SPARC expression
SPARC Expression after Treatment with MS-275 & 5-AZC Larson et al 2010Slide11
Treatment with both MS-275 & 5-AZC for 72hrsConclusion: No change in SPARC expressionSPARC Expression after Drug Combination Treatment
Larson
et al
2010Slide12
SPARC Expression after Treatment with As⁺³ & Cd⁺²
Larson
et al
2010Slide13
SPARC Expression during Transformation of UROtsa Cell LinesSlide14
Summary: SPARC ExpressionModerate SPARC expression in Normal TissueLow or undetectable SPARC expression in Cancerous/Tumor Tissue
SPARC expression does not appear to be regulated epigenetically in our system
SPARC mRNA and protein expression decreases with increasing concentrations of As
3+
and Cd
2+Slide15
SPARC Transfection4 cell lines were chosen for stable transfection of SPARC: As#3, As#6, Cd#1, Cd#4
All cell lines were characterized:
mRNA
Protein
Secretion of SPARC protein
Growth
MigrationSlide16
SPARC Transfection4 cell lines were chosen for stable transfection of SPARC: As#3, As#6, Cd#1, Cd#4Slide17
All cells were grown in Serum Free growth mediaMedia was harvested, centrifuged, and filteredProtein was precipitated from growth media
Loaded equal total protein per lane
SPARC Expression within Cultured Media
SPARC
transfected
cell lines had similar staining
SPARC expression does not appear to be matrix incorporatedSlide18
Growth: MTT
Cell Line
Doubling Time
Parent
33.2 ± 0.8 h
As
#
3
33.3 ± 1.4 h
As
#
3-SPARC
27.4 ± 1.0 h*
As
#
6
21.6 ± 1.6 h
As
#
6-SPARC
25.6 ± 0.5 h
Cd
#
1
27.8 ± 0.6 h
Cd
#
1-SPARC
23.8 ± 0.6 h*
Cd
#
4
20.7 ± 1.1 h
Cd
#
4-SPARC
22.0 ± 0.6 h
Cao
et al
2010;
Somji
et al
2010Slide19
Wound/Scratch assay:
Migration
Cells were grown to confluence
Conditions:
Untx
or treated with
Mitomycin
C (MMC) for 2hScratch performed with 200ul pipette tipCells were rinsed with PBS 2x’sPictures taken at 0h and 24hMTT performed to determine cell viabilityDetermine best concentration of MCC
to prevent cell death Performed in duplicateSlide20
Wound/Scratch assay: MigrationSlide21
MigrationTranswell Migration assay:
Top chamber = 2.4 x 10
5
cells/ml in serum free media
Bottom chamber = 1.5% fetal calf serum
8h incubation for migration
Count total number of cells on insert
Remove cells from top of insert and count remainingTotal of 20 fields per insert, performed in duplicateSlide22
MigrationTranswell Migration assay:
**
**
*
*
*
*
*Slide23
ConclusionsExpression of SPARC is not epigenetically regulated in our systemSPARC expression decreased with treatment with Cd2+ & As
3+
SPARC
tranfections
:
Secrete SPARC into media
2
transfected cell lines had decreased migrationSlide24
Future DirectionsAssess the ability of SPARC transfected cell lines to form tumors in nude miceTumor size
Subcutaneous vs.
intraperitoneal
tumor formation
Expression of SPARC in tumor
Assess the structural differences of SPARC between UROtsa Parent and transfected cell lines
Glycosylation
Post-translational modificationsCharacterization of the promoter elements that turn off SPARC expression with treatment of Cd2+ & As3+Slide25
AcknowledgementsDr Jane DunlevyHolly HewittDr Don Sens
Dr Mary Ann
Sens
Dr
Seema
Somji
Dr Scott GarrettDr XuDong ZhouSlide26
Questions?Slide27Slide28Slide29
SPARCFirst identified as the most abundant non-collagenous component of bone (Termine et al.1981)
Highly conserved among different species
Human gene has 92% homology with mice
Predicted molecular mass of 32,511 Daltons
Secreted form of the protein migrates at 43kD on SDS-PAGE
Due to addition of carbohydrates
(Sage et al. 1984)
Bradshaw & Sage 2001Slide30
SPARC Expression in Mouse Heterotransplants
Larson
et al
2010Slide31
SPARC Expression in Cancerous TissueTumor Promoter:Increased expression of SPARCBrain: Glioblastomas,
Astrocytomas
, Meningioma
Breast
: Invasive ductal carcinoma
Lung
: NSCLC, squamous cell carcinoma, adenocarcinoma
Pancreas: Pancreatic ductal adenocarcinomaSkin: MelanomaTumor Suppressor:Decreased expression of SPARCBrain: NeuroblastomaOvary: carcinomaLung: NSCLC & SCLCPancreas: PDACSkin
: MelanomaUterus: Cervical & endometrial carcinomaSlide32
Epigenetic Regulation of SPARCOvarian cancer:
Treatment with the
demethylating
agent 5-aza-2'-deoxycytidine (5-AZC) rescued SPARC mRNA and protein expression in 9 ovarian cancer cell lines
1 cell line showed a significant increase in SPARC mRNA after
TSA
treatment,
histone deacetylase inhibitor (Socha et al. 2009)Colon cancer:Induction of SPARC was observed in 5 of the 7 cell lines after 5-AZC treatment (Yang et al. 2007)Non-small cell lung cancer (NSCLC):SPARC expression was restored after 5-AZC treatment in all 11 NSCLC cell lines (Suzuki et al. 2005)
Pancreatic adenocarcinoma:SPARC mRNA expression was restored in seven 7 of 8 cell lines after 5-AZC treatment SPARC expression was not restored 1 cell line after 5-AZC treatment did not restore the SPARC expression. Treatment with an HDAC inhibitor TSA or with a combination of 5-AZC and TSA did not induce SPARC expression (Sato et al. 2003)